Mechanisms of phospholipase A_2-dependent killing of microorganisms on macrophages

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The purpose of the present study is to investigate the profiles of intracellular translocation and phosphorylation of cytosolic phospholipase A_2(cPLA_2) in macrophage (Mφ) infected with Mycobacterium tuberculosis (MTB) or Mycobacterium avium complex (MAC). We also examined the mode of intramacrophage signal transduction related to the cPLA_2 translocation and phosphorylation. The present study gave the following findings, (1)ATP significantly potentiated Mφ anti-MTB or anti-MAC bactericidal activity. This effects of ATP was specifically blocked by a cPLA_2 inhibitor, arachidonyl trifluoromethylketone (a-TFMK). (2)Intramacrophage translocation of membranous arachidonic acid (AA) molecules to MAC-containing phagosomes was also specifically blocked by a-TFMK. (3)By confocal microscopic observation of MAC-infected Mφs, it was found that ATP enhanced intracellular translocation of cPLA_2 into MAC-containing phagosomes. (4)This cPLA_2 translocation was found to coincide with infection-induced Mφ apoptosis. (5)When CHO cells and CD36^+CHO cells transfected with GFP tagged cPLA_2 (GFP-cPLA_2) were infected with MAC, intracellular GFP-cPLA_2 was observed to translocate to the MAC-containing phagosomes. Since cellular cPLA_2 activities and translocation are tightly regulated by phosphorylation of certain serine residues of cPLA_2 molecules, it is of interest to examine the roles of cPLA_2's serine residues, including Ser505,Ser515 and Ser727,in intracellular translocation and activation cPLA_2 in MAC-infected Mφs. In this context, we are currently attempting to generate vectors encoding the cDNAs of GFP-tagged cPLA_2 with appropriate phosphorylation site mutations.